The invention relates generally to cells of the immune system and more specifically to clonal amplification of lymphocytes.
Higher animals are distinguished by the presence of an immune system which serves to recognize and respond to foreign materials, or antigens, invading the organism. These functions are carried out by cells called lymphocytes which exist in two classes, B lymphocytes or B-cells and T lymphocytes or T-cell. When activated by an antigen, B-cells differentiate and secrete antibodies, or immunoglobulins, which recognize and bind to foreign substances. Activated T-cells perform a variety of functions including assisting particular B-cells and initiating reactions to eliminate the antigen.
Individual B-cells and T-cells are both highly specific to particular antigens. Each B-cell produces a single homogeneous antibody species, distinguished by the amino acid sequence of its peptide chains, which can bind to only a very limited array of antigens. T-cells derive their specificity from the structure of antibody-like molecules located on their surfaces, termed T-cell receptors, or TCRs, which also recognize and bind a limited array antigens.
Mammals typically possess 100,000 to 100,000,000 lymphocytes of different specificities, collectively capable of responding to a vast range of antigens. Most lymphocytes in human peripheral blood are nondividing. Some, however, are dividing because mature antigen-reactive T-cells and B-cells periodically undergo clonal amplification whereby a cell having specificity for a particular antigen divides repeatedly to provide a large number of identical cells or clones. Clonal amplification of lymphocytes can occur in response to specific antigens or other stimuli. Antigenic stimuli may derive from external stimuli such as infectious agents or organ transplants, or from endogenous stimuli such as tissue antigens in autoimmune diseases or neoantigens in cancer. By generating large numbers of immunologically reactive cells, this clonal amplification may either benefit the host, by promoting resistance to infection or rejection of tumors, or may in fact harm the host, by causing autoimmune diseases or the rejection of organ transplants. Clonal amplification can also occur in lymphomas, such as leukemia, where malignant lymphocytes divide repeatedly.
T-lymphocytes acquire their unique antigen specificity early in the life of the individual as they differentiate in the thymus. Antibodies and TCRs are composed of multiple peptide chains: the heavy and light chains in antibodies and the .alpha., .beta., etc., chains in the TCRs, all encoded by the corresponding genes. Within each of these genes are multiple related nucleotide sequences encoding the so-called variable (V), constant (C), diversity (D) and joining (J) regions. During maturation of the lymphocytes, these DNA regions are separately rearranged in each cell to give a particular combination of C and V, D and J regions and confer the uniqueness of the antigen specific receptors found in mature reactive T- and B- cells. Each gene rearranges independently in a cell to yield a great potential diversity of specific antigen reactive cells marked by a like diversity of TCR gene DNA restriction fragment patterns. There may be 25 to 50 copies of the V region, and several copies of each of the other regions, providing some millions of possible unique rearrangements. Once rearranged, the gene patterns remain throughout the life of the mature lymphocyte and in all of its clonal descendants. Because these cells presumably persist for many years, the patterns endure for the life of the individual.
The ability to recognize lymphocyte clonal amplification in vivo would have enormous diagnostic and therapeutic benefits. For example, the early diagnosis of premalignancy is often crucial for implementing effective therapy. Identifying a clonal amplification, even one resulting in a vast number of clonal cells, is particularly difficult because of the large potential diversity of unique antigen sensitive cells. A large clonal amplification is merely a needle in the haystack within the vast repertoire of antigen-sensitive cells. A method that would selectively direct attention to those lymphocytes that are actively dividing or have recently divided in vivo, would greatly facilitate such identification. The present invention satisfies these needs and provides related advantages as well.